Electron microscope with raman spectroscopy

ABSTRACT

Electron microscope provided, in the direction of the longitudinal axis, with at least one electron beam generation system, a condenser and objective lens system, a specimen chamber with a specimen mount, a projection lens system with imaging screen for the purpose of transmission electron microscopy (TEM) and/or an electron detector for the purpose of scanning electron microscopy (SEM) . The microscope is used in combination with an externally positioned Raman spectrometer and an associated light source for injecting and extracting, via a window in the microscope wall, a light beam to be directed at the specimen, and specimen-related Raman radiation, respectively. In the specimen chamber, a light beam and Raman radiation guide system is provided with an optical guide to guide the light beam to--and the Raman radiation from--the specimen. The guide system and the specimen mount are displaceable with respect to one another for mutual alignment of the specimen and the optical axis of the Raman spectrometer.

FIELD OF THE INVENTION

The invention relates to an electron microscope provided, in thedirection of the longitudinal axis, with at least one electron beamgeneration system, a condenser and objective lens system, a specimenchamber with a specimen mount, a projection lens system with an imagingscreen for the purpose of transmission electron microscopy (TEM) and/oran electron detector for the purpose of scanning electron microscopy(SEM), for use in combination with an externally positioned Ramanspectrometer and an associated light source for injecting andextracting, via a window in the microscope wall, a light beam to bedirected at the specimen, and specimen-related Raman radiation,respectively. Such a microscope is disclosed by the French PatentApplication FR-A-2596863.

BACKGROUND OF THE INVENTION

The electron microscope generally known from the prior art is aninstrument by means of which it is possible for structures in thinspecimens or surfaces to be rendered visible, for example by means ofTEM, or for structures in bulk specimens to be rendered visible, forexample by means of SEM, with high resolving power, in the order ofmagnitude of a few tenths of nanometres in the former case or of a fewnanometres in the latter case.

In such a microscope, as depicted in FIG. 1, an electron beam 1 isproduced in an electron gun 2 consisting of cathode, Wehnelt cylinderand anode. With the aid of a magnetic lens system 3, a so-calledcondenser lens system, the electron beam is focused into a coherent spotabove the specimen 4, which spot may be subjected to a scanning notion.

In the case of the transmission electron microscope (TEM) the beampasses through the specimen 4 and is projected with the aid of amagnetic lens system, such as objective 5 and projection lenses 6, ontoan imaging screen 7 such as a fluorescent screen. Upon incidence of theelectron beam, this screen lights up and produces a magnified image ofthe specimen. The presence, in the specimen, of elements having varyingatomic numbers results in contrast being achieved, heavy elements havinga high atomic number affecting the electron trajectory in a differentway than do light elements having a lower atomic number. The result ofthis is that, at positions where heavy elements are present, theelectrons are reflected in their entirety or in part, while electronscan pass through fairly easily at positions where light elements arepresent. Consequently, a specimen composed of different chemicalelements will therefore also transmit varying quantities of electrons.On the screen an image is formed which gives the highest brightness forpositions where light elements having lower atomic numbers are presentin the specimen, and the lowest brightness at positions where elementshaving a high atomic number are present. A sort of shadow image is thusformed.

In the case of the scanning electron microscope (SEM) which is depictedin FIG. 2 and which is essentially of the same construction as thetransmission electron microscope, the electron beam 1 generated by anelectron gun 2 also passes through a condenser lens system 3. Theelectron beam, in the case of the SEM, is focused onto a spot on thespecimen, said spot being subjected to a scanning motion by means of adeflection unit 10. The electrons reflected or backscattered by thespecimen 4 are intercepted at an electron detector 8 and, afteramplification in 11, are used to effect intensity variations on thescreen of a cathode ray tube 12 synchronized with the electron beamscanning.

The TEM provides images of thin specimen cuts and can therefore rendervisible the interior of specimens to be studied. In contrast, the SEMprovides an image with the aid of the electrons returning from thespecimen and is therefore specifically suitable for presenting images ofthe surface, or directly below it, of a specimen. If the specimen to bestudied is a section through an object, the SEM will naturally alsoprovide information on the interior. It is also possible, in the SEM,for the reflected electrons to be detected selectively. Thus we candistinguish between secondary electrons and backscattered electrons. Thenumber of electrons which is detected as backscattered may be a measurefor the chemical elements which are present in the specimen. Thus anelement having a high atomic number will reflect more electrons than anelement having a low atomic number, a difference in brightness thusbeing produced, which is representative for the elemental composition ofthe specimen.

Another possibility to obtain elemental information with the aid of theelectron microscope is the use of Electron Energy Loss Spectrometry(EELS). This technique is used in transmission electron microscopy andis based on the principle that electrons are slowed down in the specimenand that the degree of slowing down depends on the density (elementalcomposition) of the specimen. As a result of use being made of a type ofelectron prism, the electrons, after having been slowed down by thespecimen are deflected differently causing so-called element-specificimages to be formed.

For that matter, there are also so-called scanning-transmission electronmicroscopes (STEM), in which the TEM and the SEM systems are combinedand their imaging options are connected. In this case it will bepossible, via two separate monitors, to observe both the TEM image andthe SEM image.

Each of the abovementioned electron microscopes may in additionincorporate an X-ray detector. By making use of the X-rays which arereleased as a result of the beam-specimen interaction, it is possible,by means of this detector, to obtain information concerning the presencein the specimen of certain chemical elements. As is known, incidentprimary electrons from the electron beam may collide with the electrons,present in the specimen, of atoms. As a result of this collision, theelectrons which are located in one of the innermost shells of the atomsmaking up the specimen, may be knocked out of their orbit, thusproducing an unstable atom. To eliminate this instability, an electronfrom a high orbit having a particular energy level can drop back to saidlow orbit, energy being released in the process which in part is emittedin the form of X-radiation and is specific for the element in question.

FIG. 3 indicates how the electron beam 1, after passing through thecondenser aperture 13 and the pole pieces 14 impinges on the specimenplaced on a grid holder 15. Part of the electron beam passes through thespecimen and an objective lens 16. The X-radiation 18 released from thespecimen is intercepted above the specimen in an X-ray detector 17 andcan, when an image point is being investigated, be represented in theform of a spectrum on a monitor (see FIG. 4) or can, when an entirepicture element is being studied, be represented in the form of anelement distribution picture on another monitor.

By focusing, as stated earlier, the electron beam into a scanning spoton that part of the specimen which is to be studied, it is possible togenerate the X-radiation for that specific part of the specimen. Theresolving power of said X-ray microscopy is determined by the diameterof the electron beam and is in the order of magnitude of from 0.05 to0.5 μm. The spectra obtained with the aid of said X-ray microanalysismainly provide information on the presence of chemical elements in thespecimen, but do not provide information on the structure of moleculesand/or crystals in situ. It is indeed possible, with the TEM, to obtaininformation with respect to the occurrence of specific crystals, fromthe diffraction pattern which can be produced with a TEM. With a SEM,however, a diffraction pattern cannot be obtained, and the knowledge theinvestigator in question has of the specimen will determine whether heis able to derive, to some extent, the molecular and crystal structurefrom the elemental composition.

From a different optical spectroscopic technique, then, Ramanspectroscopy is known, which involves irradiating a specimen withmonochromatic light. The light scattered by the specimen will, inaddition to light of the same wavelength as the incident light, alsocontain light of other wavelengths. This wavelength shift is caused byan interaction between molecules of the specimen and photons of theincident light, which results in the molecules, after interaction,remaining in a vibrational energy state which is different from theinitial one. Different molecular vibrations lead to different discretewavelength shifts. Thus information is obtained with respect to themolecular composition and molecular structure of the specimen.

A Raman microspectrometer consists of an optical microscope, opticallycoupled to a spectrometer provided with a sensitive optical detector(photomultiplier, cooled CCD camera). The spatial resolution which canbe achieved with such an instrument is determined by the opticaldiffraction limit (i.e. ˜λ/2). The Raman photon energy collected in theoptical detector may serve to form a spectrum as depicted in FIG. 5,when an image point is studied, or to display a distribution picturewhen an entire image element is studied.

FIG. 6 depicts a highly schematic design of a Raman spectroscope, whichis used to carry out the abovementioned spectrometry. 20 indicates alight source such as a laser, 21 a laser premonochromator, 22 a lens andcondenser system, 23 a beam splitter, 24 an objective, 25 a specimen, 26a prism, 27 an ND filter, 28 a camera, 29 a monitor, 30 a spatialfilter, 31 a lens, 32 a Raman spectrometer, for example aphotomultiplier, 33 a control unit and 34 an x-y plotter. This Ramanspectroscope can be operated in two modes when the specimen 25 isstudied. In the one, or imaging mode, it is possible to present on themonitor 29, via the camera, a distribution picture of the informationfrom the specimen in the spectrum for specific Raman components. In theother, or analysis mode, a Raman spectrum of small areas or image pointsof the specimen can be studied by means of the spectrometer 32.

A drawback of said known Raman spectroscopy is the relatively widewavelength spectrum and the large spot size of the light beam with whichthe specimen could be irradiated. As a result of the use of microlasertechniques, the resolving power of Raman spectrometry has now beengreatly improved. In addition, work is currently being done on thedevelopment of Raman spectrometry in combination with Scanning NearField Microscopy (SNOM). In this application the light required togenerate the Raman signal is radiated in via a nanotip of a hollow tubeor fibre whose terminal piece has a smaller cross section than thewavelength of the light used. As a result, the specimen is "illuminated"by an extremely thin light beam which provides for a resolving powerwhich is below the theoretical value of the resolving power of a normaloptical microscope.

The abovementioned electron microscopy and Raman spectroscopy aregenerally carried out separate from one another. If the information fromthese two techniques has to be related, it is extraordinarily difficultto synchronize the results of these techniques. In the meantime there isa great need, both in material science and in medical-biologicalresearch, for identifying molecules and crystals in electron microscopicspecimens.

The abovementioned French patent application discloses the use of anelectron microscope in combination with an externally positioned Ramanspectrometer and associated light source. The light beam of the lightsource is injected into the microscope via a window in the microscopewall at the level of the condenser and objective lens system and isreflected directly at 45°, via a reflection plate positioned on thelongitudinal axis of the electron microscope, in the direction along thelongitudinal axis of the electron microscope, and is directed at thespecimen. The Raman radiation subsequently emanating from the specimenis deflected via the same reflection plate and is extracted, via thewindow, towards the Raman spectrometer. In practice it proved mostdifficult thus to achieve alternating or virtually simultaneousoperation of electron microscopy and Raman spectrometry. The combinationof a light-optical system and an electron-optical system, simultaneouslypresent in the space above the specimen compartment around the electronlongitudinal axis, quite quickly causes deformation of the electronbeam, for example astigmatism. Furthermore, the space between the poleshoes and the specimen is limited, to the extent that the introductionof the light-optical system is disadvantageous, in particular in thecase of transmission electron microscopy. This limited space alsopresents problems in positioning the X-ray analysis detector.

SUMMARY OF THE INVENTION

The object of the invention is to overcome the abovementioned problemsand to further develop an electron microscope in such a way that anelectron microscope is obtained which is easy to handle and isefficiently combined with Raman spectrometry, in which electronmicroscope the results of the two analysis techniques are coordinatedeffectively.

This is achieved, according to the invention, in an electron microscopeof the type mentioned in the preamble, in that, in the specimen chamber,a light beam and Raman radiation guide system is provided to guide thelight beam to--and the Raman radiation from--the specimen, and in thatthe guide system and the specimen mount are displaceable with respect toone another for mutual alignment of the specimen and the optical axis ofthe Raman spectrometer.

With this embodiment according to the invention it was found,surprisingly, that as a result of making use of modern techniques ingenerating light beams of very small diameter and high intensity the useof Raman spectroscopy in terms of resolving power is highly expedient inelectron microscopy.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be explained in more detail by means of a specificembodiment with reference to the drawings, in which:

FIG. 1 shows a schematic diagram of a known transmission electronmicroscope (TEM);

FIG. 2 shows a schematic diagram of a known scanning electron microscope(SEM);

FIG. 3 shows a detail rendition of a specimen chamber with an X-raymicroanalysis detector;

FIG. 4 shows an example of an X-ray microanalysis spectrum of an imagepoint;

FIG. 5 shows an example of a Raman spectrum of an image point;

FIG. 6 shows a schematic diagram of a known Raman spectroscope;

FIG. 7 shows, by way of a sketch, a schematic diagram of a specificembodiment according to the invention in a transmission electronmicroscope;

FIG. 8 shows a more detailed elaboration of the specific embodiment fromFIG. 7;

FIG. 9 shows, by way of a sketch, a schematic diagram of anotherspecific embodiment according to the invention in a scanning electronmicroscope; and

FIG. 10 shows a more detailed elaboration of the specific embodimentfrom FIG. 9.

DETAILED DESCRIPTION OF THE INVENTION

In FIG. 7, 40 schematically indicates a transmission electronmicroscope. In this electron microscope, 41 indicates an electron gun,42 a condenser lens system, 43 an objective lens, 45 generally aspecimen chamber, 44 a specimen mount, 46 an intermediate and projectionlens system, 48 an imaging screen, 47 a CCD camera, and 49 and 49.1 incombination a Raman microanalysis system with injection and extractionmechanism.

FIG. 8 depicts the combination of components 49 and 49.1 from FIG. 7 indetail, 50 indicating a light source such as a microlaser, 50.1 apremonochromator, 50.2 a semi-transparent prism, 51 in general a lenssystem, 52 pole shoes, 53 a specimen displacement mechanism, 54 a lightbeam and Raman radiation guide system provided with an objective andlens system 54.1 and an optical guide 54.2, for guiding and injectingthe light beam towards the specimen and for guiding and extracting theRaman signals emanating from the specimen, and 55 a Ramanmicrospectrometer. The light beam and Raman radiation guide system 54can additionally be provided with a second objective and lens system54.3 and a light guide 54.4 for guiding and extracting the Raman signalsemanating from the specimen to the additionally providedsemi-transparent prism 50.3.

This system can be combined with an x-y plotter 58 and/or with acomputer 56 which contains an image memory, so that theelectron-microscopic image can be read in with the aid of a CCD camera57. Subsequently, a cursor in this image may serve to allow the user toorient himself and to guide and indicate the positioning of the specimenin the optical longitudinal axis of the Raman microspectroscope.

In FIG. 9, which shows an example of an embodiment in the scanningelectron microscope, 60 schematically indicates a scanning electronmicroscope. In this electron microscope, 61 indicates an electron gun,62 a condenser lens system, 63 a beam deflector controlled by a scanninggenerator 63.1, 65 in general a specimen chamber, 64 a specimen mount,69 an electron or X-ray detector, 68 an amplifier, 66 an imaging screen,74 an image computer, 67 and 67.1 in combination a Raman microanalysissystem with injection and extraction mechanism.

FIG. 10 depicts the combination of components 67 and 67.1 from FIG. 9 indetail, 79 indicating a light source such as a microlaser, 79.1 apremonochromator, 79.2 a semi-transparent prism, 70 in general anobjective and lens system, 71 a specimen displacement mechanism, 76 ingeneral a lens system, 75 a specimen, 77 pole shoes, 70 a light beam andRaman radiation guide system provided with an objective and lens system70.1 and an optical guide 70.2, for guiding and injecting the laserlight towards the specimen 75 and for guiding and extracting the Ramansignals emanating from the specimen, and 73 a Raman microspectrometer.This system can be combined with an x-y plotter 80 and/or with acomputer 74 which contains an image memory, so that theelectron-microscopic image can be read in with the aid of a CCD camera78. Subsequently, a cursor in this image may serve to allow the user toorient himself and to guide and indicate the positioning of the specimenin the optical longitudinal axis of the Raman microspectroscope.

It goes without saying that, although this is not indicated, it ispossible for both types of microscopes indicated in the FIGS. 8 and 10to be fitted with a microlaser and Raman signal detector of the Ramanspectroscope embodied inside the microscope per se, and/or to be fittedwith an X-ray microanalysis detector, as in FIG. 3.

In the abovementioned specific embodiments in FIGS. 8 and 10, the laserand Raman guide system has been positioned next to the microscope axisin an adjustable manner, it being possible for the specimen to beseparately aligned therewith. In both examples, the specimen mount 53 or71 is movable, in a controllable manner from the outside, between thepositions A and B, which makes it possible for measurements with theelectron detector (and X-ray detector) in position A to be carried outalternately with measurements with the Raman spectroscope in position B.In the latter position, the laser beam is injected via the optical guideor fibre and is radiated in, parallel to the longitudinal axis of themicroscope, onto the specimen, the Raman radiation emanating from thespecimen being extracted. With the design according to the invention ina transmission electron microscope according to FIG. 8 it is alsopossible, when studying thin specimens, for the Raman radiation emergingat the underside of the specimen to be collected and extracted via thelens 54.3 and the optical guide 54.4.

In both the abovementioned specific embodiments from the FIGS. 8 and 10it is also possible, instead of displacing the specimen between thepositions A and B, for the light beam and Raman radiation guide system54 or 70 to be displaced, owing to the flexible connection with thelight guide, to the position A directly above the specimen. The specimenis then irradiated by the light beam from the space directly below thepole shoes 52 or 77.

It is further possible, when near field microscopy with a nanotip 72 atthe end of the light guide is used, rather than displacing the specimenbetween the positions A and B, to displace the flexible light guide 54.2or 70.2 to the position A directly above the specimen. With this design,having a nanotip 72 at the light guide, an objective and lens system54.1 or 70.1 is unnecessary.

The light beams which can be generated with modern microlasers can havea high intensity with a very narrow bandwidth. At the same time, becausethe cross-section of the laser beam can be made small. Ramanmicrospectroscopy consequently, in terms of resolving power, willapproach X-ray microanalysis. Use of the SNOM in combination with theRaman microspectrometer even permits a resolving power which is belowthe theoretically achievable resolving power of an optical microscope.

The invention can advantageously be applied both in material science andin biology. With, for example, the manufacture of implants for bonetissue it is important to characterize the apatite crystals present inthe inorganic matrix of bone tissue, and the crystals in calciumphosphate ceramics which are used as bone implants. In addition it isimportant to establish, in laboratory animal experiments, whether, andif so, to what extent, changes in the composition of the two matricesoccur during the implantation period. Likewise, it is then advantageousto establish the type of possible degradation products produced bycellular disintegration.

With the aid of the abovementioned design according to the invention itis possible, to considerable advantage, for (portions of) a specimen tobe studied accurately by simultaneous observation of the electronmicroscopy picture on a first monitor and the Ramanspectrum/spectroscopy picture on a second monitor, supplemented, ifrequired, by the X-ray analysis spectrum/X-ray analysis picture on athird monitor. Since the X-ray analysis provides elemental information,and Raman analysis provides molecular information, the two can becombined in a surprising manner, which makes it possible for both theX-ray signal and the Raman signal separately to be interpreted moreeffectively, the two enhancing one another.

Given the specific embodiments indicated above, the number ofapplications to be expected for molecular-identification systems in anelectron microscope is extraordinarily large.

We claim:
 1. In an electron microscope (40; 60) provided, in thedirection of a longitudinal axis, with at least one electron beamgeneration system (41; 61), a condenser and objective lens systemcomprising a plurality of elements (42, 43; 62, 62), a specimen chamber(45; 65) with a specimen mount (44; 64), a projection lens system (46)with imaging screen (48) for the purpose of transmission electronmicroscopy (TEM) and/or an electron detector (69) for the purpose ofscanning electron microscopy (SEM), for use in combination with anexternally positioned light source (50; 79) and associated Ramanspectrometer (55; 73) with a light beam and Raman radiation guide systemfor injecting, via a window in a microscope wall, a light beam to bedirected at the specimen respectively for extracting via said window ofspecimen-related Raman radiation, the improvement wherein the light beamand Raman radiation guide system (49.1; 67.1) is provided within thespecimen chamber (45; 65) aside from the microscope longitudinal axis,and a specimen (53; 71) displacement mechanism is fitted to displace thespecimen mount (44; 64) from a position in the longitudinal axis of themicroscope transversely to a position aside from said longitudinal axisin which the optical axis in said guide system (49.1; 67.1) and thespecimen are aligned.
 2. Electron microscope according to claim 1,wherein the light beam and Raman radiation guide system comprises anoptical guide (54.2; 70.2) connected to the externally positioned lightsource (50; 79) and associated Raman spectrometer (55; 73).
 3. Electronmicroscope according to claim 2, wherein the light beam and Ramanradiation guide system (49.1; 67.1) further comprises an objective andlens system (54.1: 70.1) positioned at the end of the optical guide(54.2; 70.2).
 4. Electron microscope according to claim 2, wherein theoptical guide is extended such that its end is displaceable to aposition in which the optical axis running out from the end of the guidecoincides with the longitudinal axis of the microscope and is alignedwith the specimen.
 5. Electron microscope according to claim 4, whereinthe optical guide is embodied with a nanotip at its end, for the purposeof near field microscopy.
 6. Electron microscope according to claim 1,wherein an X-ray analysis detector is fitted in the specimen chamber, torecord X-rays generated in the specimen, for the purpose of X-rayanalysis.
 7. Electron microscope according to claim 1, wherein anelectron prism is fitted, beyond the specimen in the direction of thelongitudinal axis, for the purpose of electron energy loss spectrometry.